Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms.     

Regional differences in the effects of insulin-induced hypoglycemia on phospholipid metabolism of rat brain

Insulin-induced hypoglycemia in rats may lead to stimulated brain activity and if severe enough, they may develop a stupor-coma condition. In this study, the effects of insulin-induced hypoglycemia on brain phospholipid metabolism were examined in rats which were prior injected with ³²Pi. Three hours after insulin injection (1 or 5 units/100 g body wt, i.p.), there was an increase (25%) in radioactivity of the lipid phase of cerebral cortex, but radioactivity in the cerebellum tended to decrease instead. Radioactivity in the aqueous phase of cortex was not altered after insulin injection, but that in the cerebellum was decreased by 30%. Differences were observed in labeling of individual phospholipids in response to the hypoglycemic treatment. A marked decrease in labelled phosphatidate was observed in the cerebellum from the hypoglycemic samples, but not in the cerebral cortex. In the cortex, hypoglycemic condition resulted in an increase in ³²Pi uptake into the phospholipids. However, the differences in the amount of label among individual phospholipids suggest that phosphatidylinositol and phosphatidylcholine are turning over more rapidly than other phospholipids. The hypoglycemic rats also showed a 3-fold increase in the brain free fatty acid level, but the level of diacylglycerol was not changed. Results thus suggested a correlation between the free fatty acid release and the increased turnover of phosphatidylinositol and phosphatidylcholine during brain stimulation due to insulin-induced hypoglycemia.     

The luminescent bacteria toxicity test: Its potential as an in vitro alternative

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC₅₀ values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.     

Cryopreservation of isolated rat hepatocytes

Summary Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b₅ and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b₅ and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism. This work was supported by Grant CA-30241 from the National Institutes of Health, Bethesda, Maryland.     

Activation of arachidonoyl-phosphatidylinositol and phosphatidylcholine turnover by K+-evoked stimulation of brain synaptosomes

The turnover of arachidonoyl groups in synaptosomal phospholipids after stimulation by K⁺ was examined. Raising the K⁺ concentration in the incubation medium from 5 to 55 mM caused a rapid hydrolysis of labeled arachidonate from the synaptosomal phospholipids. Under this condition, radioactivity released from phosphatidylinositols was proportionally higher than that from phosphatidylcholines. Hydrolysis of arachidonoyl group from phospholipids was correlated to an increase in radioactivity in the free fatty acid-ion complex which appeared in the interphase after extraction with chloroform-methanol 2:1 (v/v). The K⁺-evoked phospholipid hydrolysis and the formation of fatty acid-ion complex, were Ca²⁺-dependent. Phospholipid deacylation activity was localized mainly in synaptic vesicles and synaptic plasma membranes but not in the mitochondria. The stimulated turnover of synaptosomal phospholipids appeared to be mediated by the deacylation-reacylation mechanism, because similar treatment with high K⁺ stimulated the incorporation of labeled arachidonate into phosphatidylinositols and phosphatidylcholines of synaptosomes. The possible physiological implication of membrane lipid involvement in synaptic processes is discussed.     

Azido analogs of acridine: Photoaffinity probes for frameshift mutagenesis in Salmonella typhimurium

In order to identify a photoaffinity probe for 9-aminoacridine frameshift mutagenesis, 20 azido analogs of acridine were synthesized and tested in Ames' Salmonella tester strains, TA1535, TA1537, TA1538 and their corresponding excision-repair-proficient strains TA1975, TA1977, and TA1978, to determine their mutagenicity and toxicity relative to 9-aminoacridine. The substituent-mutagenicity patterns observed for these compounds agree very well with those obtained previously for non-azidoacridines. The results presented here show that the 2-azido-analog of 9-aminoacridine demonstrates biological activity similar to 9-aminoacridine prior to photolytic activation. With light activation, however, the 9-amino-2-azido derivative becomes more effective at producing frameshift mutations characteristics of 9-aminoacridine. Furthemore, this photolytic enhancement of mutagenesis appears to be due to the repairable lesion suggesting that covalent attachment of the drug occurs.     

The Entire Compound Autosomes of DROSOPHILA MELANOGASTER

Three new unusual compound chromosomes have been synthesized in Drosophila melanogaster. They consist of two homologous autosomes joined together in the new order: right arm, left arm, centromere, left arm, right arm, for each of the two major autosomes, and one in which chromosomes 2 and 3 have been combined in the order: right arm of 2, left arm of 2, centromere, left arm of 3, right arm of 3. The attachments of the autosomal arms were accomplished by obtaining chromosome breaks at or very close to the ends of the left arms of the autosomes such that no essential chromosome material has been removed; the compounds derived from them are therefore referred to as entire compounds. These large chromosomes are recovered in progeny with frequencies lower than expectation partly because of zygote mortality associated with these chromosomes, and partly because of a failure of spermiogenesis.     

Rapid effects of BCG on T cell function in cancer patients

Summary Forty-four cancer patients were evaluated with multiple sequential tests to determine the effects of a single intracutaneous injection of BCG on the levels of total and high-affinity sheep erythrocyte-forming cells and on lymphoproliferative responses to a T cell mitogen (PHA) and antigens (MLC and PPD). The analysis of the data was complicated by considerable pretreatment variation in the results with some patients and by the lack of a consistent change in responses among all patients. However, exploratory statistical procedures made it possible to demonstrate that inoculation with BCG was associated with a significant transient increase, on day 3 post-injection, in levels of total and active rosette-forming cells and in lymphoproliferative responses to PPD. The effects on responses to PHA varied with the level of pretreatment reactivity with patients with initial low responses showing a significant increase on days 3 and 7 and patients with initial normal responses showing a significant decrease on days 3 and 7. Patients and normal individuals receiving no injection, and patients receiving isotonic saline injections, showed none of these changes. Thus it appears that BCG may cause measurable changes in T cell levels and lymphoproliferation to T cell mitogen (PHA) and antigen (PPD).     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.